The most common method for detection of eluted species in liquid chromatography is to measure the ultraviolet absorbance in a flow-through cell located downstream of the column outlet. If any component in the mobile phase has a significant absorbance at the detection wavelength, this will give rise to a non-zero baseline in the chromatogram, which can however be simply offset if the baseline is constant. If the column is eluted with a mobile phase gradient where one component in the gradient shows UV absorbance, an increasing or decreasing baseline will be formed, for which correction methods have been suggested in e.g. EP299652A1.
A more complex situation often occurs in the chromatography of proteins and other biomacromolecules, where aqueous buffers are normally used as mobile phases and it is common to vary the pH and/or the ionic strength during elution. Many buffer components, in particular carboxylic acids, have different UV spectra for the acid and the base form and thus the UV absorbance varies with pH and also with the ionic strength, which affects the dissociation equilibria. This gives rise to baseline variations which are not simply monotonic and there is thus a need for correction of these variations to simplify the evaluation of the chromatograms.